Clostridium difficile is a spore-forming anaerobic Gram-positive bacillus that causes gastrointestinal illnesses in humans ranging from mild diarrhea to pseudomembranous colitis, which in severe cases can lead to toxic megacolon, an acute and sometimes lethal form of colonic distension. The incidence, severity and mortality of C. difficile colitis have increased significantly over the last two decades. However, knowledge concerning interactions between C. difficile bacteria and the human host is virtually nonexistent, severely impeding the development of new approaches to the prevention, control, and treatment of C. difficile disease. Type IV pili are fimbrial surface appendages produced by many bacteria that play critical roles in cellular adhesion, colonization, twitching motility, biofilm formation,and horizontal gene transfer. They are often essential for virulence and some have been successfully developed as vaccines. Type IV pili have been characterized extensively in Gram-negative bacteria, but nearly nothing is known about these pili from Gram-positive bacteria. Type IV pilin genes, though, are present in the genomes of all members of the genus Clostridium and all C. difficile strains encode complete sets of T4P biogenesis machinery components and a variable number of Type IV pilin proteins. We hypothesize that C. difficile Type IV pili, through structural features distinct from those of Gram-negative Type IV pili, directy mediate human cell adherence that is important for both colonization and virulence. Accordingly, we seek to define C. difficile Type IV pilin structures, both as individual protein components and assembled supramolecular appendages, and to identify their human host cell receptors, by pursuing the following Specific Aims: (1) to determine the atomic structures of individual C. difficile Type IV pilin proteins; (2) to define the composition and architecture of C. difficile Tye IV pili; and (3) to identify host molecules engaged specifically by C. difficile Type IV pili. The comprehensive approach that we propose to investigate the structure and function of C. difficile Type IV pili will utilize protein structure determination methods throughout a broad range of resolutions, including X-ray crystallography, NMR spectroscopy, small-angle X-ray scattering and cryo-electron microscopy, as well as state-of-the-art mass spectrometry-based approaches to define the composition of multi-component protein assemblies, map protein-protein interfaces and identify novel receptor molecules. To assure the success of our proposed studies, we have assembled a team of outstanding investigators with extensive expertise in these experimental techniques, C. difficile biology and Type IV pilus structure and function.